Project Summary Gene expression is controlled by transcription factors that are often bound to chromatin hundreds of kilobases away. This long-range control of gene expression is accomplished through chromatin interactions as part of the 3D organization of the genome. Genome-wide studies of chromatin interactions (Hi-C) have identified several patterns of chromatin organization including compartments, topologically associated domains (TADs), and high intensity point-to-point loops. Fundamental principles causing the formation of these structures are not understood. For example, human cells form high intensity loops that are associated with CTCF and are dependent on its motif?s orientation. A loop extrusion model has been proposed to explain this feature of mammalian CTCF motif orientation, but the mechanistic principle behind this phenomenon is unknown. I found that in Drosophila, CTCF does not form high intensity loops and the motif orientation does not correlate with any interaction preferences. Instead, non-CTCF loops occur in early development, but disappear in later stages when compartments and TADs appear. In C. elegans, which lacks CTCF altogether, high intensity loops correspond to dosage compensation complex recruitment sites. My preliminary work in analyzing C. elegans data indicates that a network of these loops spans the X chromosome. One major goal of this project is to discover fundamental principles of loop formation. Aim1 of this project will A) exploit the differences between CTCF looping in humans and Drosophila to determine how human CTCF loops form, B) explore the relationship of loops to TADs and compartments throughout development, and C) test whether or not loops create a network of interactions that contributes to the overall X chromosome structure and gene expression control in C. elegans. My previous work has indicated that transcription plays an important role in compartment and TAD formation, a feature that can be somewhat obscured by the prominent role of architectural proteins, like CTCF. Aim2 of this project will confirm the role of transcriptional elongation in chromatin organization and will decipher the individual roles of architectural proteins and transcriptional activity in compartment and TAD formation. These findings will contribute to refining a preliminary algorithm that can simulate chromatin organization at high resolution, giving researchers the ability to predict the effects of mutations or chromatin aberrations on TAD structure. The experiments within these aims will be initiated during the K99 phase of the award and will include training on genetic methods and the considerations necessary for working with Drosophila and C. elegans. This training will provide me with the tools and mentorship necessary for a successful transition to independent research during the R00 phase.